Fatty acids are one of the most extensively studied classes of compounds due to their important role in biological systems (Ferrante, A., Hii, C. S. T., Huang, Z. H., Rathjen, D. A. In The Neutrophils New Outlook for the Old Cells. (Ed. Gabrilovich, D.) Imperial College Press (1999) 4: 79-150; Sinclair, A., and Gibson, R. (eds) 1992. Invited papers from the Third International Congress. American Oil Chemists'Society, Champaign, Ill. 1-482). Hundreds of different fatty acids exist in nature and among them. Naturally occurring polyunsaturated fatty acids (PUFAs) contain 16 to 22 carbon atoms with two or more methylene-interrupted double bonds.
PUFAs can be divided into four families, based on the parent fatty acids from which they are derived: linoleic acid (18: 2 n-6), α-linolenic acid (18: 3 n-3), oleic acid (18: 1 n-9) and palmitoleic acid (16: 1 n-7). The n-6 and n-3 PUFAs cannot be synthesized by mammals and are known as essential fatty acids (EFAs). They are acquired by mammalian bodies indirectly through desaturation or elongation of linoleic and α-linolenic acids, which must be supplied in the diet.
EFAs have a variety of biological activities and n-3 PUFAs are required for normal human health (Spector, A. A. (1999) Lipids 34, 1-3). For instance, dietary n-3 PUFAs have effects on diverse physiological processes impacting normal health and chronic disease (for a review, see, for example, Jump, D. B. (2002) J. Biol. Chem. 277, 8755-8758), such as the regulation of plasma lipid levels (Rambjor, G. S., Walen, A. I., Windsor, S. L., and Harris, W. S. (1996) Lipid 31, 45-49; Harris, W. S. (1997) Am. J. Clin. Nutr. 65, 1645-1654; Harris, W. S. Hustvedt, B-E., Hagen, E., Green, M. H., Lu, G. and Drevon, C. A. (1997) J. Lipid Res. 38, 503-515; Mori, T. A., Burke, V., Puddey, I. B., Watts, G. F., O'Neal, D. N., Best, J. D., and Beilen, L. J. (2000) Am. J. Clin. Nutr. 71, 1085-1094), cardiovascular (Nordoy, A. (1999) Lipids 34, 19-22; Sellmayer, A., Hrboticky, N., and Weber, P. C. (1999) Lipids 34, 13-18; Leaf, A. (2001) J. Nutr. Health Aging 5, 173-178) and immune function (Hwang, D. (2000) Annu. Rev. Nutr. 20, 431-456), insulin action (Storlien, L., Hulbert, A. J., and Else, P. L. (1998) Curr. Opin. Clin. Nutr. Metab. Care 1, 559-563; Storlien, L. H., Kriketos, A. D., Calvert, G. D., Baur, L. A., and Jenkins, A. B. (1997) Prostaglandins Leukotrienes Essent. Fatty Acids 57, 379-385), and neuronal development and visual function (Salem, N., Jr., Litman, B., Kim, H-Y., and Gawrisch, K. (2001) Lipids 36, 945-959). Ingestion of n-3 PUFA will lead to their distribution to virtually every cell in the body with effects on membrane composition and function, eicosanoid synthesis, and signaling as well as the regulation of gene expression (Salem, N., Jr., Litman, B., Kim, H-Y., and Gawrisch, K. (2001) Lipids 36, 945-959; Jump, D. B., and Clarke, S. D. (1999) Annu. Rev. Nutr. 19, 63-90; Duplus, E., Glorian, M., and Forest, C. (2000) 275, 30749-30752; Dubois, R. N., Abramson, S. B., Crofford, L., Gupta, R. A., Simon, L. S., Van De Putte, L. B. A., and Lipsky, P. E. (1998) FASEB J. 12, 1063-1073).
Additionally, it has been suggested that n-3 PUFAs are important modulators of neoplastic development because they are capable of decreasing the size and number of tumours as well as the lag time of tumour appearance (Abel, S., Gelderblom, W. C. A., Smuts, C. M., Kruger M. (1997) Pros. Leuko. and Essential, 56 (1): 29-39). Intake of n-3 PUFAs has been found to be associated with a reduced incidence of coronary arterial diseases, and various mechanisms by which n-3 PUFAs act have been proposed (Krombout, D. (1992) Nutr. Rev. 50: 49-53; Kinsella, J. E., Lokesh, B., Stone R. A. (1990) Am. J. Clin. Nuer. 52: 1-28). Some n-3 PUFAs also possess antimalarial (Kumaratilake, L. M., Robinson, B. S., Ferrante, A., Poulos A. (1992) J. Am. Soc. Clin. Investigation 89: 961-967) or anti-inflammatory properties (Weber, P. C. (1990) Biochem. Soc. Trans. 18: 1045-1049).
Furthermore, one of the EFAs' most important biological roles is to supply precursors for the production of bioactive fatty acid metabolites that can modulate many functions (Arm, J. P., and Lee, T. H. (1993) Clin. Sci. 84: 501-510). For instance, arachidonic acid (AA; 20:4, n-6) is metabolized by Cytochrome P450 (CYP) enzymes to several classes of oxygenated metabolites with potent biological activities (Roman R J. P-450 metabolites of arachidonic acid in the control of cardiovascular function. Physiol Rev. 2002; 82:131-85). Major metabolites include 20-hydroxyeicosatetraenoic acid (20-HETE) and a series of regio- and stereoisomeric epoxyeicosatrienoic acids (EETs). CYP4A and CYP4F isoforms produce 20-HETE and CYP2C and CYP2J isoforms EETs.
It is known that EPA (20:5, n-3) may serve as an alternative substrate for AA-metabolizing CYP isoforms (Theuer J, Shagdarsuren E, Muller D N, Kaergel E, Honeck H, Park J K, Fiebeler A, Dechend R, Haller H, Luft F C, Schunck W H. Inducible NOS inhibition, eicosapentaenoic acid supplementation, and angiotensin II-induced renal damage. Kidney Int. 2005; 67:248-58; Schwarz D, Kisselev P, Ericksen S S, Szklarz G D, Chemogolov A, Honeck H, Schunck W H, Roots I. Arachidonic and eicosapentaenoic acid metabolism by human CYP1A1: highly stereoselective formation of 17(R),18(S)-epoxycicosatetraenoic acid. Biochem Pharmacol. 2004; 67:1445-57; Schwarz D, Kisselev P, Chemogolov A, Schunck W H, Roots I. Human CYP1A1 variants lead to differential eicosapentaenoic acid metabolite patterns. Biochem Biophys Res Commun. 2005; 336:779-83; Lauterbach B, Barbosa-Sicard E, Wang M H, Honeck H, Kargel E, Theuer J, Schwartzman M L, Haller H, Luft F C, Gollasch M, Schunck W H. Cytochrome P450-dependent eicosapentaenoic acid metabolites are novel BK channel activators. Hypertension. 2002; 39:609-13; Barbosa-Sicard E, Markovic M, Honeck H, Christ B, Muller D N, Schunck W H. Eicosapentaenoic acid metabolism by cytochrome P450 enzymes of the CYP2C subfamily. Biochem Biophys Res Commun. 2005; 329:1275-81). A remarkable feature of CYP-dependent n-3 PUFA metabolism is the prefrerred epoxidation of the n-3 double bond which distinguishes EPA and DHA from AA. The resulting metabolites—17,18-EETeTr from EPA and 19,20-EDP from DHA—are unique in having no homolog within the series of AA products.
EETs and 20-HETE play important roles in the regulation of various cardiovascular functions (Roman R J. P-450 metabolites of arachidonic acid in the control of cardiovascular function. Physiol Rev. 2002; 82:131-85). It has been shown that Ang II-induced hypertension is associated with a down-regulation of CYP-dependent AA metabolism (Kaergel E, Muller D N, Honeck H, Theuer J, Shagdarsuren E, Mullally A, Luft F C, Schunck W H. P450-dependent arachidonic acid metabolism and angiotensin II-induced renal damage. Hypertension. 2002; 40:273-9) in a double-transgenic rat (dTGR) model of Ang II-induced hypertension and end-organ damage (Luft F C, Mervaala E, Muller D N, Gross V, Schmidt F, Park J K, Schmitz C, Lippoldt A, Breu V, Dechend R, Dragun D, Schneider W, Ganten D, Haller H. Hypertension-induced end-organ damage: A new transgenic approach to an old problem. Hypertension. 1999; 33:212-8). The transgenic rats harbor the human renin and angiotensinogen genes, produce Ang II locally and develop significant hypertension, myocardial infarction and albuminuria. The animals die of myocardial and renal failure before the eighth week of age. The model shows severe features of Ang II-induced inflammation. Reactive oxygen species are generated, the transcription factors NF-κB and AP-1 are activated, and genes harboring binding sites for these transcription factors are activated.
Recently, it has been shown that eicosapentaenoic acid (EPA) supplementation significantly reduced the mortality of dTGR J, Shagdarsuren E, Muller D N, Kaergel E, Honeck H, Park J K, Fiebeler A, Dechend R, Haller H, Luft F C, Schunck W H. Inducible NOS inhibition, eicosapentaenoic acid supplementation, and angiotensin II-induced renal damage. Kidney Int. 2005; 67:248-58). Additionally, it has been shown that dTGR develop ventricular arrhythmias based on Ang II-induced electrical remodeling (Fischer R, Dechend R, Gapelyuk A, Shagdarsuren E, Gruner K, Gruner A, Gratze P, Qadri F, Wellner M, Fiebeler A, Dietz R, Luft F C, Muller D N, Schirdewan A. Angiotensin II-induced sudden arrhythmic death and electrical remodeling. Am J Physiol Heart Circ Physiol. 2007; 293:H1242-1253). Treatment of the dTGR rats with a PPAR-alpha activator strongly induced CYP2C23-dependent EET production and protected against hypertension and end-organ damage (Muller D N, Theuer J, Shagdarsuren E, Kaergel E, Honeck H, Park J K, Markovic M, Barbosa-Sicard E, Dechend R, Wellner M, Kirsch T, Fiebeler A, Rothe M, Haller H, Luft F C, Schunck W H. A peroxisome proliferator-activated receptor-alpha activator induces renal CYP2C23 activity and protects from angiotensin II-induced renal injury. Am J Pathol. 2004; 164:521-32). Long-term feeding of dTGR (from week 4 to 7 of age) with a mixture of pure EPA- and DHA-ethyl esters (Omacor from Solvay Arzneimittel, Hannover, Germany) improved the electrical remodeling of the heart in this model of angiotensin II-induced hypertension. In particular, EPA and DHA reduced the mortality, suppressed the inducibility of cardiac arrhythmias and protected against connexin 43-gap junctional remodeling (Fischer R, Dechend R, Qadri F, Markovic M, Feldt S, Herse F, Park J K, Gapelyuk A, Schwarz I, Zacharzowsky U B, Plehm R, Safak E, Heuser A, Schirdewan A, Luft F C, Schunck W H, Muller D N. Dietary n-3 polyunsaturated fatty acids and direct renin inhibition improve electrical remodeling in a model of high human renin hypertension. Hypertension. 2008 February; 51(2):540-6). EPA was also shown to reduce the spontaneous beating rate, to prevent Ca2+ induced arrhythmias and to electrically stabilize neonatal rat cardiomyocytes (Leaf A, Kang J X, Xiao Y F, Billman G E. Clinical prevention of sudden cardiac death by n-3 polyunsaturated fatty acids and mechanism of prevention of arrhythmias by n-3 fish oils. Circulation. 2003; 107:2646-52). In general, CYP-dependent eicosanoids have to be considered as second messengers: EETs and 20-HETE are produced by CYP enzymes after extracellular signal induced release of AA from membrane phospholipids (by phospholipase A2) and exert their function in the context of signaling pathways modulating ion transport, cell proliferation and inflammation. Depending on the diet, n-3 PUFAs partially replace AA at the sn2-position of phospholipids and may thus become involved as alternative molecules in the subsequent signaling pathways.
The few studies on the biological activities of CYP-dependent eicosanoids in the heart indicate important roles for EETs and 20-HETE in the regulation of L-type Ca2+ and sarcolemmal and mitochondrial ATP-sensitive potassium (KATP) channels. In cardiac myocytes, L-type Ca2+ currents and cell shorting are reduced upon inhibition of EET generation and these effects can be reversed by adding 11,12-EET (Xiao Y F, Huang L, Morgan J P. Cytochrome P450: a novel system modulating Ca2+ channels and contraction in mammalian heart cells. J. Physiol. 1998; 508 (Pt 3):777-92). EETs were also shown to activate cardiac KATP channels. This effect is highly stereoselective: only the S,R but not the R,S-enantiomer of 11,12-EET was effective (Lu T, VanRollins M, Lee HC. Stereospecific activation of cardiac ATP-sensitive K(+) channels by epoxyeicosatrienoic acids: a structural determinant study. Mol. Pharmacol. 2002; 62:1076-83). Overexpression of the EET-generating human CYP2J2 resulted in an improved postischemic functional recovery of the transgenic mouse heart via activation of KATP channels (Seubert J, Yang B, Bradbury J A, Graves J, Degraff L M, Gabel S, Gooch R, Foley J, Newman J, Mao L, Rockman H A, Hammock B D, Murphy E, Zeldin D C. Enhanced postischemic functional recovery in CYP2J2 transgenic hearts involves mitochondrial ATP-sensitive K+ channels and p42/p44 MAPK pathway. Circ Res. 2004; 95:506-14). 20-HETE appears to play an opposite role by acting as an endogenous KATP channel blocker (Gross E R, Nithipatikom K, Hsu A K, Peart J N, Falck J R, Campbell W B, Gross G J. Cytochrome P450 omega-hydroxylase inhibition reduces infarct size during reperfusion via the sarcolemmal KATP channel. J Mol Cell Cardiol. 2004; 37:1245-9; Nithipatikom K, Gross E R, Endsley M P, Moore J M, Isbell M A, Falck J R, Campbell W B, Gross G J. Inhibition of cytochrome P450omega-hydroxylase: a novel endogenous cardioprotective pathway. Circ Res. 2004; 95:e65-71).
Although n-3 PUFAs play important roles in the biological process of the mammalian body, they are not widely used as therapeutics due to their limited availability in vivo. They are readily degradable by β-oxidation, which is the major oxidative pathway in fatty acid metabolism. The net process of β-oxidation is characterised by the degradation of the fatty acid carbon chain by two carbon atoms with the concomitant production of equimolar amounts of acetyl-coenzyme A.
To overcome the problem of β-oxidation, WO96/11908 discloses modified PUFAs, such as the g-oxa and (3-thia PUFAs). These compounds were shown to have enhanced resistance to β-oxidation while still retaining certain biological activities of the native PUFAs.
Finally, new agents for the treatment or prevention of conditions and diseases associated with inflammation, proliferation, hypertension, coagulation, immune function, heart failure and cardiac arrhythmias are of considerable interest as these conditions account for a significant number of death in patients and administration of many of the presently employed drugs is associated with complex drug interactions and many adverse side effects.
Therefore, the problem underlying the present invention is to provide new analogues of n-3 PUFA metabolites, which are more stable against deactivation by soluble epoxide hydrolase and/or are less prone to auto-oxidation, and which have anti-inflammatory, anti-proliferative, anti-hypertension, anti-coagulation, or immune-modulating activity, especially cardioprotective activity.